1. Field of the Invention
This invention relates to a diazo reagent, a method for producing the diazo reagent, and a total bilirubin assay employing the diazo reagent.
2. Description of the Prior Art
For years, spectrophotometric methods have been employed for the measurement of total bilirubin in serum. In these methods, bilirubin is coupled with a diazotized compound, e.g., p-diazobenzene sulfonic acid (p-DBS) to form a colored azobilirubin. The color intensity of the azobilirubin thus formed is measured spectrophotometrically.
To form the diazo reagent, an aryl amine is diazotized with a nitrite containing compound in the presence of an acid. For example, p-aminobenzene sulfonic acid is diazotized with sodium nitrite in the presence of hydrochloric acid. The resulting diazo reagent, e.g., p-DBS, is stable for only a few hours (1-4). Efforts have been made to stabilize p-DBS solutions by adding aromatic sulfonic acid (5) or the making of fluoroborate derivatives (6). These efforts have resulted in making p-DBS stable for one day at room temperature and three days when stored at 2.degree.-8.degree. C.
When a mixture nitriloaromatic sulfonic acid (7) was utilized to stabilize p-DBS solution, the stability of p-DBS improved to two days at room temperature and three months at 2.degree.-8.degree. C.
Although these approaches have improved the stability of diazo reagents, the advances achieved thereby have nevertheless been modest. Accordingly, it would be very desirable to develop a diazo reagent having even more pronounced stability.